AbstractPresenilin is thought to be the proteolytic component of γ-secretase, responsible for the intramembranous cleavage of substrates that include the activated Notch receptor. Recent studies have identified the novel protein Nicastrin as another essential component of the Presenilin/γ-secretase complex

Lai, Eric C

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Notch Cleavage: Nicastrin Helps
Presenilin Make the Final Cut
Dispatch
Eric C. Lai
Presenilin is thought to be the proteolytic compo-
nent of γ-secretase, responsible for the intramem-
branous cleavage of substrates that include the
activated Notch receptor. Recent studies have iden-
tified the novel protein Nicastrin as another essential
component of the Presenilin/γ-secretase complex.
Notch is a transmembrane receptor that is the focal
member of a widely utilized, eponymous signal trans-
duction cascade; it mediates cell–cell interactions and
pattern formation in all metazoan organisms examined
to date. Nearly a decade ago, several groups inde-
pendently noticed that a constitutively active Notch
fragment consisting of its intracellular domain (NIC) is
nuclearly localized, a provocative observation that
suggested a function for this transmembrane protein
in the nucleus (reviewed in [1]). Although the evidence
for this hypothesis took some years to accumulate, it
is now generally accepted that activation of Notch at
the cell surface by ligand triggers proteolytic cleavage
and release of NIC, which then translocates to the
nucleus and functions as a transcriptional coactivator.
Now a new player has been identified: the novel
protein Nicastrin, which recent studies [2–4] show
plays an important part in the Presenilin-mediated
cleavage and release of NIC from the plasma
membrane.
Notch Cleavage: It’s As Easy As S1, S2, S3
Notch processing is unexpectedly complex and
includes multiple proteolytic events (Figure 1) referred
to as the S1, S2 and S3 cleavages (reviewed in [5]). S1
cleavage in the extracellular domain of Notch occurs
constitutively in the trans-Golgi network and is
mediated by a furin-like convertase; reassembly of the
fragments creates a heterodimeric Notch receptor at
the cell surface. This event has been most closely
characterized with respect to mammalian Notch, but
there is evidence that fly Notch is similarly processed.
S2 cleavage by a disintegrin/metalloprotease occurs
in response to ligand binding and releases the major-
ity of the extracellular domain. This cleavage is
believed to be mediated by TACE in vertebrates and
may be mediated by the related but distinct protein
Kuzbanian in Drosophila, although the precise role of
the latter protein is controversial at present.
The resulting membrane-anchored fragment,
referred to as Notch extracellular truncation (NEXT), is
subject to intramembranous S3 cleavage by γ-secre-
tase, which finally releases NIC. The mechanism of this
particular cleavage has attracted considerable interest
for two reasons. First, it is the key event that regulates
nuclear translocation of NIC and its second life as a
transcriptional coactivator. Second, a processed
carboxy-terminal fragment of the β-amyloid precursor
protein (β-APP) is subject to a highly similar intramem-
branous cleavage, an event that underlies the forma-
tion of cytotoxic plaques found in patients afflicted
with Alzheimer’s disease. Understanding the
mechanism of γ-secretase-mediated cleavage is thus
potentially of great medical relevance as well.
Presenilin and Nicastrin in S3 Cleavage
A wealth of studies using invertebrate and vertebrate
model systems demonstrated that Presenilin is a key
component of γ-secretase which is biochemically
required for Notch S3 cleavage and genetically
essential for Notch signal transduction (reviewed in
[5]). Presenilin is an eight-transmembane domain,
putative aspartyl protease which is widely thought to
be the enzyme that cleaves γ-secretase substrates.
Presenilin co-purifies with the γ-secretase complex
and is labeled by transition-state analogue inhibitors
of γ-secretase. However, direct demonstration of
Presenilin-mediated substrate cleavage in vitro has
Current Biology, Vol. 12, R200–R202, March 19, 2002, ©2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)00749-2
545 Life Sciences Addition, University of California,
Department of Molecular and Cell Biology, Berkeley,
California 94720-3200, USA. lai@fruitfly.bdgp.berkeley.edu
Figure 1. Summary of Notch cleavage events.
S1 cleavage in the extracellular domain of Notch (red), catal-
ysed by a furin-like convertase, occurs constitutively in the
Golgi. The two halves are reassembled as an intramolecular
heterodimer present at the plasma membrane. Interaction with
ligand induces S2 cleavage by a disintegrin/metalloprotease
(TACE, and possibly Kuzbanian), generating the Notch extra-
cellular truncation (NEXT). NEXT is processed by the γ-secre-
tase complex (oval), which includes Nicastrin (blue) and
Presenilin (green). Nicastrin is a single pass transmembrane
protein that might aid in the assembly or trafficking of the γ-
secretase complex to the plasma membrane and/or help to
recruit substrates. Presenilin is an eight-transmembrane
domain, putative aspartyl protease thought to be the catalytic
component of γ-secretase. The order and timing of γ-secre-
tase/substrate assembly is not well defined, and they may exist
as a complex as Notch is trafficked to the cell surface (i.e., prior
to S2 cleavage). However, only the NEXT fragment, and not
heterodimeric Notch, is efficiently cleaved by γ-secretase. S3
cleavage in the transmembrane domain releases NIC, which
subsequently translocates to the nucleus.
S3
S2S1
NEXT NIC
Current Biology
Current Biology
R201
not yet been possible, probably because of the
requirement for additional factors in the large
~2000 kD γ-secretase complex.
In a satisfying convergence of genetic and
biochemical approaches, orthologs of a novel single-
pass transmembrane protein involved in Notch signal
transduction and γ-secretase activity were recently
isolated independently from worms and humans. This
protein, first known as APH-2, was identified from a
screen for maternal-effect mutants of the nematode
Caenorhabditis elegans that have phenotypes resem-
bling those resulting from mutations in GLP-1, one of
two nematode Notch receptors [6]. APH-2 localizes to
cell-surface membranes and is required for GLP-1-
mediated cell–cell interactions in the early embryo. It
is subsequently required for signaling by LIN-12, the
other worm Notch receptor, during development of
the somatic gonad [7].
Complementary biochemical studies [8] identified
the human homolog of APH-2, Nicastrin, as a protein
that could be stoichiometrically immunopurified with
Presenilin. Yu et al. [8] named the protein after the
Italian village Nicastro, home to members of an
extended family that were key to studies that eventu-
ally linked mutations in the two human presenilin genes
to the most frequent forms of familial Alzheimer’s
disease. Nicastrin forms a ternary complex with Prese-
nilin and the γ-secretase substrates NEXT or carboxy-
terminal β-APP [8,9]. Furthermore, misexpression of
various forms of Nicastrin was found to modulate pro-
cessing of β-APP, indicating a functional link between
Nicastrin and γ-secretase. Finally, Nicastrin function
has been conserved, as human Nicastrin can partially
complement the aph-2 mutant phenotype [7].
In spite of these compelling links between Nicastrin
and Notch signaling, some important connections
remained to be made. For example, misexpression of
various Nicastrin mutants that strongly affect β-APP
processing only mildly affect Notch processing [9].
Also, aph-2 mutations affect some, but not all settings
of Notch activity [7]. These negative data could be
reasonably explained, however, if Notch processing
was efficient enough to withstand the presence of
putative dominant-negative Nicastrin isoforms, while
maternal contributions might obscure the apparent
genetic requirements for aph-2. The Struhl, St. John-
ston and Fortini groups [2–4] have simultaneously
characterized Drosophila Nicastrin and now reported
powerful genetic evidence for its general requirement
during Notch S3 cleavage in vivo.
Ovary and post-embryonic clones of nicastrin
mutant cells in Drosophila, as well as embryos
deficient for maternal and zygotic Nicastrin, display a
panoply of Notch-like defects which encompass many
different settings of Notch activity [2–4]. These include
a failure of ovarian follicle cells to cease cell division
and begin differentiation, hyperplasia of the embry-
onic and adult nervous system, thickening of wing
veins and failure to specify the wing margin. Although
ex vivo blastomere recombination experiments
seemed to indicate that APH-2 might function in either
the signal-sending or signal-receiving cell [6], mosaic
clonal analysis demonstrated that Nicastrin functions
strictly cell autonomously in vivo, and that cells do not
require Nicastrin to send the Notch signal [2,3]. Thus,
Nicastrin is generally required for Notch signaling and
functions in signal-receiving cells in Drosophila.
To determine the site of Nicastrin function in signal
reception, these researchers took advantage of two
types of constitutively active Notch deletion variant
previously used to characterize Notch and Presenilin
function. Although these variants signal independently
of ligand in wild-type tissue, they differ in their require-
ment for Presenilin. The first, ∆ECN, resembles NEXT
in that it lacks most of the extracellular domain but
remains membrane anchored; it requires Presenilin for
S3 cleavage and access to the nucleus. The second,
NIC, is soluble, translocates freely to the nucleus, and
signals independently of Presenilin. These Notch
derivatives display identical requirements for Prese-
nilin and Nicastrin for access to the nucleus and signal
transducing activity, indicating that Nicastrin also
functions in S3 cleavage of Notch (Figure 1) [2–4].
These genetic data were substantiated with biochem-
ical data demonstrating an accumulation of NEXT in
the absence of either Nicastrin or Presenilin. Although
the cellular location of S3 cleavage has been the
subject of debate, nicastrin and presenilin follicle cell
clones accumulate NEXT at the apical membrane,
suggesting that the plasma membrane is indeed the
location of S3 cleavage [3].
Possible Functions of Nicastrin
Nicastrin exhibits independent interactions with
Presenilin and substrates, which led to the proposition
that it might help to recruit substrates to the γ-secre-
tase complex for processing by Presenilin [8,9]. Sur-
prisingly, Presenilin-dependent intramembranous
cleavage does not absolutely require any specific
sequence motif. Instead, a primary determinant
appears to be that the size of the substrate extracel-
lular domain be small [10]. As some 95% of Nicastrin
is located extracellularly, it was speculated that Nicas-
trin might help to measure the size of the extracellular
domain of potential γ-secretase substrates [2,4].
Nicastrin also appears to regulate Presenilin stabil-
ity, as Presenilin levels are strongly reduced following
prolonged reduction of Nicastrin in cell culture or in
large clones in vivo [2–4]. Nicastrin, perhaps com-
plexed to substrate, might positively influence Prese-
nilin endoproteolytic maturation or incorporation into
the complex, either of which could stabilize Presenilin.
Alternatively, Nicastrin may be required for transport
of the Presenilin/γ-secretase complex to the plasma
membrane. This possibility is suggested by the obser-
vation that the normally high level of Drosophila Pre-
senilin at the apical membrane is nearly absent in
nicastrin imaginal disc clones, although subapical Pre-
senilin persists [2]. 
Curiously, APH-2 displays the reciprocal require-
ment for nematode Presenilins SEL-12 and HOP-1 for
its normal localization to the cell surface [11], further
suggesting that the γ-secretase complex may need 
to be substantially assembled prior to trafficking 
to the cell surface. In fact, it is conceivable that Notch
normally arrives at the plasma membrane already
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associated with components of γ-secretase, as
Notch–Nicastrin and Presenilin–Notch interactions can
be detected in the secretory pathway [9,12]. A more
detailed understanding of how the γ-secretase
complex is assembled, both in terms of order of addi-
tion as well as subcellular location of interactions,
should help to resolve some of these possibilities.
Finally, it should be noted that Nicastrin has some
limited homology to aminopeptidases, but directed
tests of this enzymatic activity have not given positive
results [13].
More to RIP than Notch
The lifestyle of Notch might seem highly unusual, but
at least two other transmembrane proteins, β-APP and
the tyrosine kinase ErbB-4, have now been found to
lead highly similar lives. Both are subject to Presenilin-
mediated intramembranous cleavage and nuclear
translocation of their intracellular domains, which can
function as nuclear transcriptional modulators [14,15].
The existence of additional Presenilin/Nicastrin targets
can be inferred from the finding that nicastrin and
presenilin clones display phenotypes more severe
than those of Notch clones, including specific defects
in the spectrin cytoskeleton and cadherin adhesion
complexes [3]. The relevant targets in these settings
remain to be identified. A variety of other artificial
substrates that share the properties of being single-
pass transmembrane proteins with small extracellular
domains are also cleaved in a Presenilin- and/or
Nicastrin-dependent manner in Drosophila [2,10],
which might reflect a more general role for γ-secretase
in the scavenging and removal of degraded or
truncated membrane proteins. The relatively specific
phenotypes of presenilin and nicastrin worm and fly
mutants would seem to indicate, however, that their
essential functions encompass the processing of a
fairly limited number of substrates.
Nicastrin/Presenilin-mediated cleavage undoubt-
edly involves additional factors. One good candidate
is the newly discovered protein APH-1. Like Prese-
nilins SEL-12 and HOP-1, APH-1 is a multipass trans-
membrane protein that is required for Notch signaling
and localization of APH-2 in nematodes [11]. APH-1
has been conserved in flies and vertebrates, and we
await further functional characterization of this novel
protein. We also anticipate that proteomic approaches
may eventually allow all components of the purified γ-
secretase complex to be identified, which should
greatly aid efforts to understand the mechanism of
proteolysis by γ-secretase.
Protein cleavage within a transmembrane domain
has recently been dubbed ‘regulated intramembra-
nous proteolysis’ (RIP), and a large number of other
RIP substrates that are processed by other types of
membrane-embedded protease — collectively termed
intramembrane-cleaving proteases, or I-CliPs — have
recently been characterized (reviewed in [16,17]). An
exploding field unto itself, RIP happens in the ER, the
Golgi, and at the plasma membrane, and occurs in
organisms ranging from bacteria to humans. Thus, it
is an ancient strategy for regulating the function of
select transmembrane proteins that live a second life
elsewhere — extracellularly, cytoplasmically, or in an
intracellular compartment. The coming years will
surely see the identification of new RIP substrates and
the elucidation of the many different mechanisms for
catalysing RIP.
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Notch Cleavage: Nicastrin Helps Presenilin Make the Final Cut 

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Notch Cleavage: Nicastrin Helps Presenilin Make the Final Cut

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